Spinal muscular atrophy (SMA) is a common neurodegenerative disease that is caused by mutations in the survival of motor neuron gene (SMN), leading to reduced levels of the SMN protein in affected individuals. In SMA, motor neurons selectively degenerate, however, the mechanism of cell death and the precise role of SMN in this process are not completely understood. In this study, we apply RNA interference (RNAi) to knockdown Smn gene expression in the murine embryonal carcinoma stem cell line P19, which can be differentiated into neuronal cells. A direct effect of Smn loss on apoptotic cell death in differentiated P19 neuronal cells, and to a lesser extent in undifferentiated cells was observed. Apoptosis could be partly reversed by expression of an SMN rescue construct, was reversible by the addition of the caspase-inhibitor ZVAD-fmk and involved the cytochrome c pathway. This study shows for the first time that knockdown of SMN results in apoptosis in mammalian neuronal cells and has implications for understanding the cause of motor neuron-specific cell loss in SMA, and for identifying novel therapeutic targets for this disease.